The present invention relates to the field of liquid chromatography and, more particularly, to the field of liquid chromatography of radiolabeled compounds.
High Performance Liquid Chromatography (HPLC), or high pressure liquid chromatography as it is sometimes referred, is used to analyze a mixture of organic compounds and identify the individual components. In this technique a multi-component solution is introduced into a stream of solvent (mobile phase) flowing over a stationary phase (contained in a tubular column). Separation of different compounds results from differences in their relative affinity for the stationary and mobile phases. Usually the separated compounds are detected using infra-red or ultra-violet sensors at the exit of the column. This is a satisfactory technique for analyzing the molecular content of biological systems, but is less affective in measuring the turnover of molecules that occur during metabolic processes. For turnover measurements, a radioactive molecule is introduced to the biological system and any metabolic products resulting from reactions of the initial molecule will be radioactive also. The metabolic products of such reactions may be referred to as radiolabeled compounds. In this application, a radiation detector is placed at the exit of the column to detect the radiolabeled compounds. Many HPLC studies in biochemistry use short-lived radiotracers, with increased sensitivity being especially important because some radiolabeled compounds are present only in low concentrations, such as intermediate metabolites.
A typical HPLC radiation detector system includes a reservoir, a pump, an injector, a separation column, and a detector, which are connected in series by tubing. The reservoir contains a solvent that is continuously pumped through the column and the detector. The injector mixes the sample to be analyzed with a small amount of solvent and injects this solution into the solvent stream. As the sample passes through the separation column the various components separate due to the differences in their transport velocity and individual compounds exit the column at different times. The properties of the column are chosen for optimal separation of the compounds of interest. Small-diameter tubing carries the compound (and solvent) from the exit of the column into a detector that uses some attribute of the compound (such as its optical absorbance) to measure the relative concentration of the compound as a function of time. Assuming that the column has separated the individual compounds, each compound will be seen as a single peak in the detector output. The separation between peaks is determined by the properties of the column, solvent, compound, and pumping speed, but typically ranges from a few seconds to minutes. The width of each peak depends on these same properties, but is typically a few seconds. The ratio of the peak width to separation determines the ability to resolve the individual peaks (i.e., the compounds).
The area of each peak is proportional to the amount of the corresponding compound in the sample. A sensitive detector with good signal-to-noise ratio is desired to accurately measure small concentrations of the compounds. In practice, radiolabeled compounds and a radiation detector provide one of the most sensitive means of performing HPLC. The radiation detector is typically a piece of CsI:Tl scintillator crystal coupled to a PIN photodiode read out in current mode. The detector is placed in close proximity to the tubing, and lead shielding is placed outside of the detector/tubing. Although the volume of tubing observed by the radiation detector is small (typically 0.75 mm diameter and ˜6 cm long), the sensitivity of the detector is sufficient for many applications. For applications where higher sensitivity is desired, the tubing is coiled around the detector. This increases the length of time that the peak spends near the detector by a factor of n, where n is the number of turns of tubing, and so increases the sensitivity by a factor of n. However, increasing the length of time that the peak spends near the detector can also increase the measured width of the peak and so degrade the ability to separate peaks.
As an example, consider the performance for the conventional radiation detector in the case of a compound whose peak width is 5 seconds at the exit of the column, the solution is pumped through the system at a rate of 1 mL/minute, and the tubing is 0.75 mm diameter. In this case the liquid flows through the tubing at 3.65 cm/sec. The sensitivity of the system will be proportional to the length of time each volume element of the liquid spends in close proximity to the detector, but the width of the peak is blurred by the same amount. If the conventional radiation detector is 2 cm diameter, and if n loops of tubing are placed around its diameter, each volume element of the liquid will spend 1.7n seconds near the detector. For five loops, the detection time is 8.5 seconds, which will broaden the apparent width of the peak from 5 seconds to 10 seconds. For a 11C radiolabeled compound (half-life=20.38 minutes, mean life 29.4 minutes) with a 10 minute residence time in the HPLC column and a measurement time of 8.5 seconds (0.14 minutes), the fraction f of disintegrations that will occur near the detector is given by f=exp(−10/29.4)*[1−exp(−0.1.4/29.4)]=0.0034. It would be desirable to improve the detection fraction f.